Reactive cholangiocyte-derived ORM2 drives a pathogenic modulation of the injured biliary niche through macrophage reprogramming

Upon liver injury, altered cholangiocyte functions can lead to a process referred to as ductular reaction (DR), which is an umbrella term harboring not excessive cell proliferation, but also intricate inflammation and fibrogenesis modulation. DR has been considered as an essential hallmark and promising therapeutic target in a variety of liver diseases. However, the potentially broad implications of DR are rarely known due to the lack of effective investigational models. In this study, transcriptomic exploration methods were utilized to determine key factors resulting from injured cholangiocytes and in macrophage alterations, while multiplex immunofluorescent detection together with an in-house optimized image processing approaches were utilized to explore spatial counterparts in situ. Furthermore, co-culture systems (microchambers and liver-on-a-chip) equipped with multiple mouse strain-derived primary liver and circulating immune cells were employed to capitulate multi-cellular interactions upon biliary injury and to study mechanistic influencers. Results illustrated that Orosomucoid 2 (ORM2) was screened out as one of the most strongly induced secretory factors expressed by mouse-derived intrahepatic cholangiocytes under bile duct injury. Furthermore, ORM2-induced monocyte recruitment and potent transcriptome alterations in liver macrophages are associated with an increase of pro-inflammatory cytokine secretion and expression of cell stress-related genes. Furthermore, ORM2-activated macrophages not only exacerbated cell stress and Orm2 expression in both cholangiocytes and hepatocytes, but also promoted fibrogenesis in hepatic stellate cells. In addition, ORM2 regulated liver macrophage functions via an ITPR2-dependent calcium pathway. In conclusion, cholangiocyte-derived ORM2 induces liver macrophage reprogramming via an ITPR2-dependent calcium pathway in response to acute and chronic biliary injury, serving as a promoter of liver disease progression. This study reveals a novel mechanism for cholangiocyte-macrophage crosstalk upon liver injury, which may draw more attention to this cellular duet as a potential therapeutic target.